Isolation of cholesterol- and deoxycholate-degrading bacteria from soil samples: evidence of a common pathway.

Nineteen different steroid-degrading bacteria were isolated from soil samples by using selective media containing either cholesterol or deoxycholate as sole carbon source. Strains that assimilated cholesterol (17 COL strains) were gram-positive, belonging to the genera Gordonia, Tsukamurella, and Rhodococcus, and grew on media containing other steroids but were unable to use deoxycholate as sole carbon source. Surprisingly, some of the COL strains unable to grow using deoxycholate as sole carbon source were able to catabolize other bile salts (e.g., cholate). Conversely, strains able to grow using deoxycholate as the sole carbon source (two DOC isolates) were gram-negative, belonging to the genus Pseudomonas, and were unable to catabolize cholesterol and other sterols. COL and DOC were included into the corresponding taxonomic groups based on their morphology (cells and colonies), metabolic properties (kind of substrates that support bacterial growth), and genetic sequences (16S rDNA and rpoB). Additionally, different DOC21 Tn5 insertion mutants have been obtained. These mutants have been classified into two different groups: (1) those affected in the catabolism of bile salts but that, as wild type, can grow in other steroids and (2) those unable to grow in media containing any of the steroids tested. The identification of the insertion point of Tn5 in one of the mutants belonging to the second group (DOC21 Mut1) revealed that the gene knocked-out encodes an A-ring meta-cleavage dioxygenase needed for steroid catabolism.

Potential use of a Yersinia ruckeri O1 auxotrophic aroA mutant as a live attenuated vaccine.

The aroA gene of Yersinia ruckeri, which encodes 5-enolpyruvylshikimate 3-phosphate synthase, was insertionally inactivated with a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of Y. ruckeri 21102 O1 by means of the suicide vector pIVET8. The Y. ruckeri aroA::Kan(r) mutant was highly attenuated when inoculated intraperitoneally into rainbow trout, with a 50% lethal dose of >5 x 10(7) CFU. The mutants were not recoverable from the internal organs 48 h post-inoculation or later. The vaccination of rainbow trout with the AroA mutant as a live vaccine conferred significant protection (relative percentage survival = 90%) against the pathogenic wild-type strain of Y. ruckeri.

Microbial synthesis of poly(beta-hydroxyalkanoates) bearing phenyl groups from pseudomonas putida: chemical structure and characterization.

New poly(beta-hydroxyalkanoates) having aromatics groups (so-called PHPhAs) from a microbial origin have been characterized. These polymers were produced and accumulated as reserve materials when a beta-oxidation mutant of Pseudomonas putida U, disrupted in the gene that encodes the 3-ketoacyl-CoA thiolase (fadA), was cultured in a chemically defined medium containing different aromatic fatty acids (6-phenylhexanoic acid, 7-phenylheptanoic acid, a mixture of them, or 8-phenyloctanoic acid) as carbon sources. The polymers were extracted from the bacteria, purified and characterized by using (13)C nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC), and differential scanning calorimetry (DSC). Structural studies revealed that when 6-phenylhexanoic acid was added to the cultures, an homopolymer (poly-3-hydroxy-6-phenylhexanoate) was accumulated. The feeding with 8-phenyloctanoic acid and 7-phenylheptanoic acid leads to the formation of copolymers of the corresponding units with the n - 2 carbons formed after deacetylation, copoly(3-hydroxy-8-phenyloctanoate-3-hydroxy-6-phenylhexanoate) and copoly(3-hydroxy-7-phenylheptanoate-3-hydroxy-5-phenylvalerate), respectively. The mixture of 6-phenylhexanoic acid and 7-phenylheptanoic acid gave rise to the corresponding terpolymer, copoly(3-hydroxy-7-phenylheptanoate-3-hydroxy-6-phenylhexanoate-3-hydroxy-5-phenylvalerate). Studies on the chemical structure of these three polyesters revealed that they were true copolymers but not a mixture of homopolymers and that the different monomeric units were randomly incorporated in the macromolecular chains. Thermal behavior and molecular weight distribution were also discussed. These compounds had a dual attractive interest in function of (i) their broad use as biodegradable polymers and (ii) their possible biomedical applications.

Genetically engineered Pseudomonas: a factory of new bioplastics with broad applications.

New bioplastics containing aromatic or mixtures of aliphatic and aromatic monomers have been obtained using genetically engineered strains of Pseudomonas putida. The mutation (-) or deletion (Delta) of some of the genes involved in the beta-oxidation pathway (fadA(-), fadB(-) Delta fadA or Delta fad BA mutants) elicits a strong intracellular accumulation of unusual homo- or co-polymers that dramatically alter the morphology of these bacteria, as more than 90% of the cytoplasm is occupied by these macromolecules. The introduction of a blockade in the beta-oxidation pathway, or in other related catabolic routes, has allowed the synthesis of polymers other than those accumulated in the wild type (with regard to both monomer size and relative percentage), the accumulation of certain intermediates that are rapidly catabolized in the wild type and the accumulation in the culture broths of end catabolites that, as in the case of phenylacetic acid, phenylbutyric acid, trans-cinnamic acid or their derivatives, have important medical or pharmaceutical applications (antitumoral, analgesic, radiopotentiators, chemopreventive or antihelmintic). Furthermore, using one of these polyesters (poly 3-hydroxy-6-phenylhexanoate), we obtained polymeric microspheres that could be used as drug vehicles.

Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism of gap gene.

Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12 Staphylococcus spp. by PCR. In the present study, AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity.

Two different pathways are involved in the beta-oxidation of n-alkanoic and n-phenylalkanoic acids in Pseudomonas putida U: genetic studies and biotechnological applications.

In Pseudomonas putida U, the degradation of n-alkanoic and n-phenylalkanoic acids is carried out by two sets of beta-oxidation enzymes (betaI and betaII). Whereas the first one (called betaI) is constitutive and catalyses the degradation of n-alkanoic and n-phenylalkanoic acids very efficiently, the other one (betaII), which is only expressed when some of the genes encoding betaI enzymes are mutated, catabolizes n-phenylalkanoates (n > 4) much more slowly. Genetic studies revealed that disruption or deletion of some of the betaI genes handicaps the growth of P. putida U in media containing n-alkanoic or n-phenylalkanoic acids with an acyl moiety longer than C4. However, all these mutants regained their ability to grow in media containing n-alkanoates as a result of the induction of betaII, but they were still unable to catabolize n-phenylalkanoates completely, as the betaI-FadBA enzymes are essential for the beta-oxidation of certain n-phenylalkanoyl-CoA derivatives when they reach a critical size. Owing to the existence of the betaII system, mutants lacking betaIfadB/A are able to synthesize new poly 3-OH-n-alkanoates (PHAs) and poly 3-OH-n-phenylalkanoates (PHPhAs) efficiently. However, they are unable to degrade these polymers, becoming bioplastic overproducer mutants. The genetic and biochemical importance of these results is reported and discussed.

The phenylacetyl-CoA catabolon: a complex catabolic unit with broad biotechnological applications.

The term catabolon was introduced to define a complex functional unit integrated by different catabolic pathways, which are, or could be, co-ordinately regulated, and that catalyses the transformation of structurally related compounds into a common catabolite. The phenylacetyl-CoA catabolon encompasses all the routes involved in the transformation of styrene, 2-phenylethylamine, trans-styrylacetic acid, phenylacetaldehyde, phenylacetic acid, phenylacetyl amides, phenylacetyl esters and n-phenylalkanoic acids containing an even number of carbon atoms, into phenylacetyl-CoA. This common intermediate is subsequently catabolized through a route of convergence, the phenylacetyl-CoA catabolon core, into general metabolites. The genetic organization of this central route, the biochemical significance of the whole functional unit and its broad biotechnological applications are discussed.

Glyceraldehyde-3-phosphate dehydrogenase-encoding gene as a useful taxonomic tool for Staphylococcus spp.

The gap gene of Staphylococcus aureus, encoding glyceraldehyde-3-phosphate dehydrogenase, was used as a target to amplify a 933-bp DNA fragment by PCR with a pair of primers 26 and 25 nucleotides in length. PCR products, detected by agarose gel electrophoresis, were also amplified from 12 Staphylococcus spp. analyzed previously. Hybridization with an internal 279-bp DNA fragment probe was positive in all PCR-positive samples. No PCR products were amplified when other gram-positive and gram-negative bacterial genera were analyzed using the same pair of primers. AluI digestion of PCR-generated products gave 12 different restriction fragment length polymorphism (RFLP) patterns, one for each species analyzed. However, we could detect two intraspecies RFLP patterns in Staphylococcus epidermidis, Staphylococcus hominis, and Staphylococcus simulans which were different from the other species. An identical RFLP pattern was observed for 112 S. aureus isolates from humans, cows, and sheep. The sensitivity of the PCR assays was very high, with a detection limit for S. aureus cells of 20 CFU when cells were suspended in saline. PCR amplification of the gap gene has the potential for rapid identification of at least 12 species belonging to the genus Staphylococcus, as it is highly specific.

Phenylacetyl-coenzyme A is the true inducer of the phenylacetic acid catabolism pathway in Pseudomonas putida U.

Aerobic degradation of phenylacetic acid in Pseudomonas putida U is carried out by a central catabolism pathway (phenylacetyl-coenzyme A [CoA] catabolon core). Induction of this route was analyzed by using different mutants specifically designed for this objective. Our results revealed that the true inducer molecule is phenylacetyl-CoA and not other structurally or catabolically related aromatic compounds.

A new class of glutamate dehydrogenases (GDH). Biochemical and genetic characterization of the first member, the AMP-requiring NAD-specific GDH of Streptomyces clavuligerus.

A new class of glutamate dehydrogenase (GDH) is reported. The GDH of Streptomyces clavuligerus was purified to homogeneity and characterized. It has a native molecular mass of 1,100 kDa and exists as an alpha(6) oligomeric structure composed of 183-kDa subunits. GDH, which requires AMP as an essential activator, shows a maximal rate of catalysis in 100 mm phosphate buffer, pH 7.0, at 30 degrees C. Under these conditions, GDH displayed hyperbolic behavior toward ammonia (K(m), 33 mm) and sigmoidal responses to changes in alpha-ketoglutarate (S(0.5) 1.3 mm; n(H) 1.50) and NADH (S(0.5) 20 microm; n(H) 1.52) concentrations. Aspartate and asparagine were found to be allosteric activators. This enzyme is inhibited by an excess of NADH or NH(4)(+), by some tricarboxylic acid cycle intermediates and by ATP. This GDH seems to be a catabolic enzyme as indicated by the following: (i) it is NAD-specific; (ii) it shows a high value of K(m) for ammonia; and (iii) when S. clavuligerus was cultured in minimal medium containing glutamate as the sole source of carbon and nitrogen, a 5-fold increase in specific activity of GDH was detected compared with cultures provided with glycerol and ammonia. GDH has 1,651 amino acids, and it is encoded by a DNA fragment of 4,953 base pairs (gdh gene). It shows strong sequence similarity to proteins encoded by unidentified open reading frames present in the genomes of species belonging to the genera Mycobacterium, Rickettsia, Pseudomonas, Vibrio, Shewanella, and Caulobacter, suggesting that it has a broad distribution. The GDH of S. clavuligerus is the first member of a class of GDHs included in a subfamily of GDHs (large GDHs) whose catalytic requirements and evolutionary implications are described and discussed.

From a short amino acidic sequence to the complete gene.

A useful strategy directed to the isolation of a required gene with a high GC content is reported. Using a degenerate oligonucleotide probe, deduced from the amino terminus of a protein, it is possible to obtain a fragment of DNA containing its encoding gene by PCR amplification. Furthermore, the cloning of a desired gene can be accomplished in two steps by using an oligonucleotide deduced (i) from an internal sequence, (ii) from a consensus sequence, or (iii) from a DNA sequence adjacent to a disrupting element (transposon, insertion sequence, cassette). This method, which could be applied to a bacteriophage, plasmid, or cosmid genomic library, has been successfully used for cloning several genes from different biological systems.

A major secreted elastase is essential for pathogenicity of Aeromonas hydrophila.

Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal hemorrhagic septicemia in rainbow trout. A gene encoding an elastolytic activity, ahyB, was cloned from Aeromonas hydrophila AG2 into pUC18 and expressed in Escherichia coli and in the nonproteolytic species Aeromonas salmonicida subsp. masoucida. Nucleotide sequence analysis of the ahyB gene revealed an open reading frame of 1,764 nucleotides with coding capacity for a 588-amino-acid protein with a molecular weight of 62,728. The first 13 N-terminal amino acids of the purified protease completely match those deduced from DNA sequence starting at AAG (Lys-184). This finding indicated that AhyB is synthesized as a preproprotein with a 19-amino-acid signal peptide, a 164-amino-acid N-terminal propeptide, and a 405-amino-acid intermediate which is further processed into a mature protease and a C-terminal propeptide. The protease hydrolyzed casein and elastin and showed a high sequence similarity to other metalloproteases, especially with the mature form of the Pseudomonas aeruginosa elastase (52% identity), Helicobacter pylori zinc metalloprotease (61% identity), or proteases from several species of Vibrio (52 to 53% identity). The gene ahyB was insertionally inactivated, and the construct was used to create an isogenic ahyB mutant of A. hydrophila. These first reports of a defined mutation in an extracellular protease of A. hydrophila demonstrate an important role in pathogenesis.

Novel biodegradable aromatic plastics from a bacterial source. Genetic and biochemical studies on a route of the phenylacetyl-coa catabolon.

Novel biodegradable bacterial plastics, made up of units of 3-hydroxy-n-phenylalkanoic acids, are accumulated intracellularly by Pseudomonas putida U due to the existence in this bacterium of (i) an acyl-CoA synthetase (encoded by the fadD gene) that activates the aryl-precursors; (ii) a beta-oxidation pathway that affords 3-OH-aryl-CoAs, and (iii) a polymerization-depolymerization system (encoded in the pha locus) integrated by two polymerases (PhaC1 and PhaC2) and a depolymerase (PhaZ). The complete assimilation of these compounds requires two additional routes that specifically catabolize the phenylacetyl-CoA or the benzoyl-CoA generated from these polyesters through beta-oxidation. Genetic studies have allowed the cloning, sequencing, and disruption of the genes included in the pha locus (phaC1, phaC2, and phaZ) as well as those related to the biosynthesis of precursors (fadD) or to the catabolism of their derivatives (acuA, fadA, and paa genes). Additional experiments showed that the blockade of either fadD or phaC1 hindered the synthesis and accumulation of plastic polymers. Disruption of phaC2 reduced the quantity of stored polymers by two-thirds. The blockade of phaZ hampered the mobilization of the polymer and decreased its production. Mutations in the paa genes, encoding the phenylacetic acid catabolic enzymes, did not affect the synthesis or catabolism of polymers containing either 3-hydroxyaliphatic acids or 3-hydroxy-n-phenylalkanoic acids with an odd number of carbon atoms as monomers, whereas the production of polyesters containing units of 3-hydroxy-n-phenylalkanoic acids with an even number of carbon atoms was greatly reduced in these bacteria. Yield-improving studies revealed that mutants defective in the glyoxylic acid cycle (isocitrate lyase(-)) or in the beta-oxidation pathway (fadA), stored a higher amount of plastic polymers (1.4- and 2-fold, respectively), suggesting that genetic manipulation of these pathways could be useful for isolating overproducer strains. The analysis of the organization and function of the pha locus and its relationship with the core of the phenylacetyl-CoA catabolon is reported and discussed.

Catabolism of phenylacetic acid in Escherichia coli. Characterization of a new aerobic hybrid pathway.

The paa cluster of Escherichia coli W involved in the aerobic catabolism of phenylacetic acid (PA) has been cloned and sequenced. It was shown to map at min 31.0 of the chromosome at the right end of the mao region responsible for the transformation of 2-phenylethylamine into PA. The 14 paa genes are organized in three transcription units: paaZ and paaABCDEFGHIJK, encoding catabolic genes; and paaXY, containing the paaX regulatory gene. The paaK gene codes for a phenylacetyl-CoA ligase that catalyzes the activation of PA to phenylacetyl-CoA (PA-CoA). The paaABCDE gene products, which may constitute a multicomponent oxygenase, are involved in PA-CoA hydroxylation. The PaaZ protein appears to catalyze the third enzymatic step, with the paaFGHIJ gene products, which show significant similarity to fatty acid beta-oxidation enzymes, likely involved in further mineralization to Krebs cycle intermediates. Three promoters, Pz, Pa, and Px, driven the expression of genes paaZ, paaABCDEFGHIJK, and paaX, respectively, have been identified. The Pa promoter is negatively controlled by the paaX gene product. As PA-CoA is the true inducer, PaaX becomes the first regulator of an aromatic catabolic pathway that responds to a CoA derivative. The aerobic catabolism of PA in E. coli represents a novel hybrid pathway that could be a widespread way of PA catabolism in bacteria.

Elucidation of conditions allowing conversion of penicillin G and other penicillins to deacetoxycephalosporins by resting cells and extracts of Streptomyces clavuligerus NP1.

Using resting cells and extracts of Streptomyces clavuligerus NP1, we have been able to convert penicillin G (benzylpenicillin) to deacetoxycephalosporin G. Conversion was achieved by increasing by 45x the concentration of FeSO4 (1.8 mM) and doubling the concentration of alpha-ketoglutarate (1.28 mM) as compared with standard conditions used for the normal cell-free conversion of penicillin N to deacetoxycephalosporin C. ATP, MgSO4, KCl, and DTT, important in cell-free expansion of penicillin N, did not play a significant role in the ring expansion of penicillin G by resting cells or cell-free extracts. When these conditions were used with 14 other penicillins, ring expansion was achieved in all cases.

Molecular characterization of the phenylacetic acid catabolic pathway in Pseudomonas putida U: the phenylacetyl-CoA catabolon.

Fourteen different genes included in a DNA fragment of 18 kb are involved in the aerobic degradation of phenylacetic acid by Pseudomonas putida U. This catabolic pathway appears to be organized in three contiguous operons that contain the following functional units: (i) a transport system, (ii) a phenylacetic acid activating enzyme, (iii) a ring-hydroxylation complex, (iv) a ring-opening protein, (v) a beta-oxidation-like system, and (vi) two regulatory genes. This pathway constitutes the common part (core) of a complex functional unit (catabolon) integrated by several routes that catalyze the transformation of structurally related molecules into a common intermediate (phenylacetyl-CoA).

The phenylacetic acid uptake system of Aspergillus nidulans is under a creA-independent model of catabolic repression which seems to be mediated by acetyl-CoA.

The filamentous fungus Aspergillus nidulans is able to grow on phenylacetic acid (PhAc) as the sole carbon source and has a highly specific phenylacetic acid transport system mediating the uptake of this aromatic compound. This transport system is also able to transport some phenoxyacetic acid (PhOAc), although less efficiently. Maximal uptake rates were observed at 37 degrees C in 50 mM phosphate buffer (pH 7.0). Under these conditions, uptake was linear for at least 1 minute, with K(m) values for PhAc and PhOAc of 74 and 425 microM, respectively. The PhAc transport system is strongly induced by PhAc and, to a lesser extent by PhOAc and other phenyl derivatives. The utilization of glucose (and other sugars), glycerol or acetate results in a substantially reduced uptake. This negative effect caused by certain carbon sources is independent of the creA gene, the regulatory gene mediating carbon catabolite repression. Negative regulation by acetate is prevented by a loss-of-function mutation in the gene encoding acetyl-CoA synthetase, strongly suggesting that this regulation is mediated by the intracellular pool of acetyl-CoA.

Molecular cloning and expression in different microbes of the DNA encoding Pseudomonas putida U phenylacetyl-CoA ligase. Use of this gene to improve the rate of benzylpenicillin biosynthesis in Penicillium chrysogenum.

The gene encoding phenylacetyl-CoA ligase (pcl), the first enzyme of the pathway involved in the aerobic catabolism of phenylacetic acid in Pseudomonas putida U, has been cloned, sequenced, and expressed in two different microbes. In both, the primary structure of the protein was studied, and after genetic manipulation, different recombinant proteins were analyzed. The pcl gene, which was isolated from P. putida U by mutagenesis with the transposon Tn5, encodes a 48-kDa protein corresponding to the phenylacetyl-CoA ligase previously purified by us (Martínez-Blanco, H., Reglero, A. Rodríguez-Aparicio, L. B., and Luengo, J. M. (1990) J. Biol. Chem. 265, 7084-7090). Expression of the pcl gene in Escherichia coli leads to the appearance of this enzymatic activity, and cloning and expression of a 10.5-kb DNA fragment containing this gene confer this bacterium with the ability to grow in chemically defined medium containing phenylacetic acid as the sole carbon source. The appearance of phenylacetyl-CoA ligase activity in all of the strains of the fungus Penicillium chrysogenum transformed with a construction bearing this gene was directly related to a significant increase in the quantities of benzylpenicillin accumulated in the broths (between 1.8- and 2.2-fold higher), indicating that expression of this bacterial gene (pcl) helps to increase the pool of a direct biosynthetic precursor, phenylacetyl-CoA. This report describes the sequence of a phenylacetyl-CoA ligase for the first time and provides direct evidence that the expression in P. chrysogenum of a heterologous protein (involved in the catabolism of a penicillin precursor) is a useful strategy for improving the biosynthetic machinery of this fungus.

Enzymatic synthesis of hydrophobic penicillins.

Our knowledge of the enzymes and genes involved in the biosynthesis of beta-lactam antibiotics has increased notably in the last decade. The purification to homogeneity of some of these proteins as well as their biochemical characterization has allowed some of them to be used for synthesizing many different penicillins and cephalosporin-like products in vitro. In this report we describe the most important advances in this field, placing special emphasis on the enzymatic synthesis of hydrophobic penicillins. The use of purified acyl-CoA: 6-aminopenicillanic acid (6-APA) acyltransferase (AT) from Penicillium chrysogenum and several acyl-CoA ligases obtained from different microbial origins has led to the reproduction "in vitro" of the last step involved in in penicillin biosynthesis. By coupling these enzymatic systems (AT and acyl-CoA ligases) an impressive number of beta-lactam antibiotics has been obtained. Thus, most of the known natural penicillins, many of the semisynthetic variants and others, which until now can only be obtained chemically, have been synthesized enzymatically from their natural precursors. Furthermore, the use of heterologous proteins in coupled systems has opened a new and exciting field in beta-lactam antibiotic research, lending new perspectives to the traditional methodology followed by antibiotic fermentation industries.